In this renewal application we extend previous studies on cell-cell interactions for both normal melanocytes and malignant melanoma cells. The laboratory has recently made two significant discoveries: i. We have found neural crest-like stem cells in the human dermis that differentiate into bona fide melanocytes which home to the epidermis, suggesting that they can serve as a continuous reservoir for epidermal pigment-producing melanocytes; ii. We discovered that Notch activation in pigment-producing melanocytes leads to de-differentiation towards a neural crest-like stem cell without malignant transformation, underscoring the plasticity of melanocytes. Our first working hypothesis is that dermal stem cell self-renewal is regulated through Notch and non-canonical Wnt signaling and is dependent on cellular localization in the context of the normal human dermis. Our second hypothesis is that melanocyte differentiation of dermal stem cells is regulated through Wnt signaling, in which signals change from the non-canonical to the canonical pathway. In the first aim we will define self-renewal of dermal neural crest-like stem cells, postulating that self-renewal is regulated through Wnt- and Notch-mediated signaling between stem cells and mesenchymal-like niche cells. We expect that Notch and Wnt signaling pathways cooperate to maintain stem cell self-renewal, and our studies are expected to reveal the mechanisms of pathway interactions. The striking similarities between dermal neural crest- like stem cells and melanoma cells underscore the significance of de-differentiation processes that cells undergo during transformation. In the second aim we will address differentiation of stem cells to melanocytes and the relocation of cells from the dermal to the epidermal skin compartment. For these studies we have developed a model of organotypic skin that allows investigations on the dynamic changes in neural crest-like stem cells as they separate from the niche to migrate through the basement membrane zone to the epidermis. During differentiation to melanocytes cells undergo profound phenotypic changes, thus understanding how these changes are induced may allow us to pursue the differentiation of malignant cells as a novel therapeutic strategy for melanoma. PUBLIC HEALTH RELEVANCE: We have discovered a new stem cell in human skin that has many properties in common with malignant cells. These stem cells are located in the lower compartment of the skin, the dermis, and can change to become melanocytes, neuronal cells, fat cells, bone cells or cartilage cells. We plan to study how they can be maintained in the skin, what it takes for them to migrate to the upper compartment of the skin, the epidermis, and what triggers their change to pigment-producing melanocytes. The stem cells share many molecules in common with melanoma cells but not with normal melanocytes, suggesting that melanoma cells should be compared to the stem cells and not the melanocytes. As we learn more about the stem cells, we begin to better understand how tumor cells migrate and invade through tissues.